Cloning and expression of a puromycin N-acetyl transferase gene from Streptomyces alboniger in Streptomyces lividans and Escherichia coli.

A gene (pac) encoding a puromycin N-acetyl transferase (PAC) of Streptomyces alboniger ATCC12461 was cloned in the Streptomyces plasmid pIJ702 and expressed in S. lividans 1326. Several clones resistant to puromycin were isolated and shown to carry pIJ702 with different inserts of S. alboniger DNA. They were classified as of low and high activity according to the levels of enzymatic activity expressed by them. The different levels of expression were related to the two possible orientations of the S. alboniger DNA inserts in the pIJ702 vector. Six of the recombinant plasmids contain a common 1.6-kb DNA sequence which, by subcloning experiments, was shown to carry a pac gene encoding PAC activity. The pac gene was subcloned next to the lac promoter of Escherichia coli plasmid pUC19. Only one of the two possible orientations of insertion expressed PAC activity, suggesting that it was dependent on the lac promoter. Accordingly, isopropylthio-beta-D-galactoside (IPTG) was able to stimulate the expression of the enzyme activity. These results allowed the direction of transcription of the pac gene to be determined.